Of an estimated 4.3 million live births in the United States each year, approximately 87 000 (about 2.1%) occur very prematurely, defined as gestational age less than 32 weeks. In Europe it is estimated that the incidence of preterm birth less than 32 weeks of gestation is 1.2% in 10 000 inhabitants. Thus with 450 million inhabitants in EU25 the incidence of preterm births less 32 weeks of gestation is expected to be 54 000 infants per year. Preterm labor and its complications are major perinatal public health issues in developed societies today. Infants with low birth-weight or infants born prematurely miss part or all of the critical period of in utero growth. They account for half of all infant deaths and three-quarters of long-term morbidity. They impose a heavy burden on the national economy, because of the high costs of special care in both the neonatal period and over the life-span of survivors. Many survivors also have diminished quality of life because of physical damage resulting directly from prematurity.
The length of a normal pregnancy or gestation is considered to be 40 weeks (280 days) from the date of conception. Infants born before 37 weeks of gestation are considered premature and may be at risk for complications. Infants born before 32 completed weeks of gestation are considered very preterm and infants born before 28 completed weeks of gestation are considered born extremely preterm. Advances in medical technology have made it possible for infants born as young as 23 weeks gestational age (17 weeks premature) to survive. Infants born prematurely are at higher risk for death or serious complications due to their low birth weight and the immaturity of their body systems. Low birth weight, defined by a cut-off of 2500 g, serves as a marker for high risk newborns, as it is correlated with prenatal risk factors, intrapartum complications and neonatal disease, and is composed largely of preterm births. Studies on very low birth weight, defined as less than 1500 g or less than 1000 g cut-offs that identify infants at highest risk, those with high rates of severe respiratory and neurological complications associated with extreme prematurity.
The lungs, digestive system, and nervous system (including the brain) are underdeveloped in premature babies, and are particularly vulnerable to complications. The most prevalent medical problems encountered in preterm infants are developmental delay, mental retardation, bronchopulmonary dysplasia, intraventricular hemorrhage and retinopathy of prematurity. When preterm children are deprived of their natural environment they lose important factors normally found in utero, such as proteins, growth factors and cytokines. It has been demonstrated that insulin-like growth factor 1 (IGF-I) is one such factor, but it is likely there are others.
Insulin growth factor I (IGF-I) is a well-known regulator of postnatal growth and metabolism. It has a molecular weight of approximately 7.5 kilo Daltons (kDa). IGF-I has been implicated in the actions of various other growth factors, since treatment of cells with such growth factors leads to increased production of IGF-I. However, its role in prenatal growth and development has only recently been recognized. Experimental data obtained in IGF-I−/− mice suggest that IGF-I play an important role in the third trimester of embryonic growth and development of several tissues. In support of the IGF-I−/− data in mice, a patient homozygous for a gene defect in the IGF-I gene was shown to display impaired prenatal growth and development of the central nervous system.
IGF-I has insulin-like activities and is mitogenic (stimulate cell division) and/or is trophic (promote recovery/survival) for cells in neural, muscular, reproductive, skeletal and other tissues. Unlike most growth factors, IGF is present in substantial quantity in the circulation, but only a very small fraction of this IGF is free in the circulation or in other body fluids. Most circulating IGF is bound to the IGF-binding protein IGFBP-3. IGF-I may be measured in blood serum to diagnose abnormal growth-related conditions, e.g., pituitary gigantism, acromegaly, dwarfism, various growth hormone deficiencies, and the like. Although IGF-I is produced in many tissues, most circulating IGF-I is believed to be synthesized in the liver. In human fetal serum, IGF-I levels are relatively low and are positively correlated with gestational age and birth weight. Almost all IGF circulates in a non-covalently associated ternary complex composed of IGF-I, IGFBP-3, and a larger protein subunit termed the acid labile subunit (ALS). The IGF-I/IGFBP-3/ALS ternary complex is composed of equimolar amounts of each of the three components. ALS has no direct IGF binding activity and appears to bind only to the IGF-I/IGFBP-3 binary complex. The IGF-I/IGFBP-3/ALS ternary complex has a molecular weight of approximately 150 kDa. This ternary complex is thought to function in the circulation “as a reservoir and a buffer for IGF-1 preventing rapid changes in the concentration of free IGF”. It has been shown that excessive free IGF-I can down regulate the bioactivity of IGFBPs; thus, reduced IGFBP activity could also contribute to the lack of effect of high-dose IGF-I. Earlier pharmacokinetic studies in healthy children and adults have found half-life of the IGF-I/IGFBPs complex to be approximately 12 to 15 hours in plasma. IGFBP-3 is the most abundant IGF binding protein in the circulation, but at least five other distinct IGF binding proteins (IGFBPs) have been identified in various tissues and body fluids. Although these proteins bind IGFs, they each originate from separate genes and have unique amino acid sequences. Thus, the binding proteins are not merely analogs or derivatives of a common precursor. Unlike IGFBP-3, the other IGFBPs in the circulation are not saturated with IGFs. Moreover, none of the IGFBPs other than IGFBP-3 can form the 150 kDa ternary complex. The IGF-I/IGFBP-3 ratio in plasma has been used as an estimate of the availability of non protein-bound IGF-I, with an increased ratio suggesting an increased availability of free bio-active IGF-I. A higher IGF-I/IGFBP-3 ratio during the first postnatal month has been associated with higher growth velocity in moderately preterm, as compared to term infants. However conclusions from measuring the IGF-I and IGFBP-3 during the first postnatal month in infant born very and extremely preterm showed no difference in the IGF-I/IGFBP-3 ratio between healthy infants with morbidity (i.e. ROP).
IGF-I and IGFBP-3 may be purified from natural sources or produced by recombinant means. For instance, purification of IGF-I from human serum is well known in the art. Production of IGF-I by recombinant processes is shown in EP 0 128 733, published in December of 1984. IGFBP-3 may be purified from natural sources using a process such as that shown in Baxter et al. (1986, Biochem. Biophys. Res. Comm. 139:1256-1261). Alternatively, IGFBP-3 may be synthesized recombinantly as discussed in Sommer et al., pp. 715-728, MODERN CONCEPTS OF INSULIN-LIKE GROWTH FACTORS (E. M. Spencer, ed., Elsevier, N.Y., 1991). Recombinant IGFBP-3 binds IGF-I in a 1:1 molar ratio.
During fetal life these elements are introduced through placental absorption or ingestion from amniotic fluid (AF). Deprivation of such factors is likely to cause inhibition or improper stimulation of important pathways, which in the case of the eye may cause abnormal retinal vascular growth, the hallmark of retinopathy of prematurity (ROP). Understanding which factors are lost with preterm birth and evaluating their impact on the development of ROP will also have much greater implications for the growth and development of other organ systems (brain, lungs, gut, and bones). Replacing lost factors is likely to improve overall development. Therefore, research in this field is of great importance for the understanding of normal development of immature infants and for the prevention of many complications of preterm birth.
ROP is a major cause of blindness in children in the developed and developing world, despite current treatment of late-stage ROP. As developing countries provide more neonatal and maternal intensive care, the incidence of ROP is increasing. Although ablation treatment, such as laser photocoagulation or cryotherapy, of the retina reduces the incidence of blindness by 25% in those with late-stage disease, the visual outcome after treatment is often poor. Preventive therapy for ROP would clearly be preferable.
Retinal blood vessel development begins during the fourth month of gestation and is not completed until term. Therefore, infants born prematurely have incompletely vascularized retinas, with a peripheral a vascular zone, the area of which depends on the gestational age. With maturation of the infant, the resulting non-vascularized retina becomes increasingly metabolically active and hypoxic. The hypoxia-induced retinal neovascularization (NV) phase of ROP is similar to other proliferative retinopathies, such as diabetic retinopathy.
In the early 1950's studies in patients with proliferative diabetic retinopathy demonstrated that pituitary ablation resulted in total remission of the retinopathy indicating that growth hormone (GH) or some factor in the GH-axis played an important role for the development of retinopathy. GH was shown to be critical for retinopathy in a mouse model. In addition, experimental studies in a ROP mouse model demonstrated that an IGF-I receptor antagonist was found to suppress retinal neovascularization. IGF-I regulates retinal NV, at least in part, through control of vascular endothelial growth factor (VEGF) activation of p44/42 MAPK (a kinase inhibitor), establishing a hierarchical relationship between IGF-I and VEGF receptors. These studies establish a critical role for IGF-I in angiogenesis. IGF-I acts permissively to allow maximum VEGF stimulation of new vessel growth. Low levels of IGF-I inhibit vessel growth despite the presence of VEGF. Therefore, IGF-I is likely to be one of the non-hypoxia-regulating factors critical to the development of ROP (L. E. Smith, W. Shen, C. Perruzzi et al., Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor. Nat Med 5 (1999), pp. 1390-1395).
Similar to the role of VEGF, it has been shown that IGF-I is critical for the normal development of the retinal vessels (Hellström, C. Perruzzi, M. Ju et al, Low IGF-I suppresses VEGF-survival signaling in retinal endothelial cells: direct correlation with clinical retinopathy of prematurity. Proc Natl Acad Sci USA. 98 (2001), pp. 5804-5808). IGF-I levels fall below in utero levels after birth, partly due to the loss of IGF-I provided by the placenta and the amniotic fluid. It has been hypothesized that IGF-I is critical to normal retinal vascular development, and that a lack of IGF-I in the early neonatal period is associated with lack of vascular growth and with subsequent proliferative ROP. In IGF-I knockout mice (IGF-I −/−), normal retinal vascular development was examined to determine whether IGF-I is critical to normal blood vessel growth. Retinal blood vessels grow more slowly in IGF-I −/− mice than in those of normal mice, a pattern very similar to that seen in premature babies with ROP. These observations were confirmed in patients with ROP (Hellström, C. Perruzzi, M. Ju et al., Low IGF-I suppresses VEGF-survival signaling in retinal endothelial cells: direct correlation with clinical retinopathy of prematurity. Proc Natl Acad Sci USA. 98 (2001), pp. 5804-5808 and Hellström A, Engstrom E, Hard A-L, et al. Postnatal serum IGF-I deficiency is associated with retinopathy of prematurity and other complications of premature birth pediatrics, Pediatrics 2003; 112: 1016-1020).
In a previous patent application, US 2004/0053838, generally related to determining the risk of developing complications of premature birth and low birth weight and to methods for treatment of such complications, these complications are associated with low levels of IGF-I. The therapeutic approach suggested treatment of complications of prematurity by administration of IGF-I to a patient, to elevate the patient's serum levels of IGF-I to an in utero baseline level. According to one of the methods for treatment in accordance with said invention, IGF-I can be administered in a composition comprising IGF-I in combination with an additional protein that is capable of binding IGF-I and propose such binding protein to be IGF-I binding protein 3 (IGFBP-3). It is further suggested that a composition comprising equimolar amounts of IGF-I and IGFBP-3 may be used.
In U.S. Pat. No. 5,187,151 (Genentech) co-administration of IGF-I and IGFBP, is suggested in general, in a molar ratio of IGFBP-3 to IGF-I of about 0.5:1 to 3:1 (or as the molar ratio is expressed as IGF-I/IGFBP-3 throughout the present application corresponding molar ratios disclosed in U.S. Pat. No. 5,187,151 would be 1:0.5 to 1:3 when compared to the molar ratios claimed in the present invention) by subcutaneous bolus injection. They state that the mixture to be used in therapy will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (including any perceived or anticipated side or reduced anabolic effects using IGF-I alone, the particular growth defect or catabolic state to be corrected, the particular IGFBP being utilized, the site of delivery of the mixture and other factors known to the practitioners. Further methods for manufacture of IGF-I and IGFBP compositions with varying molar ratios are known from prior art. However, the beneficial effect of administration of a combination of the two components in a specific ratio range is not disclosed in the literature. In particular, there is to the best of our knowledge no teaching or indication in the prior art what IGF-I based method would be expected to provide the most efficient way of treatment of premature children running a clear risk of acquiring a handicap for the rest of their life.
Despite the ever-increasing advances in the understanding of complications of prematurity, there are no presently available effective treatments or methods of determining the risk of developing these life-threatening conditions, as premature death is still the norm.